FoxP3转染未成熟DCs来源的外泌体治疗EAE的实验研究

Immune tolerance plays an important role in the pathogenesis of multiple sclerosis (MS). Immature dendritic cells (imDC) can induce immunotolerance by producing regulatory T cell (Treg). It's important that enhanced immunotolerance of imDC to treate MS. This study aims at promoting the ability of immune tolerance of immature DC transfected by FoxP3 gene ,which can guide the synthesis of marked protein on the surface of regulatory T cell. Primarily, the experimenters will constructe adenoviral vector of excessive expression of FoxP3 and transfecte immature DC derived from mice's bone marrow. Secondly, it is crucial to extract exosomes from transfected immature DC and analyze changes of protein and miRNA before and after transfection by applying proteomics and gene chip technology. Finally, the research will take exosomes involved into in vivo and in vitro intervention experiments while observing the effect on immune inflammation and myelin regeneration. It should be note that the functions of exosomes are associated with the changes of proteins and miRNA of exosomes produced by immature DC transfected by FoxP3. The study is performed to explore the correlation between proteins or miRNA and the experimental results in vivo and in vitro, in order to reveal mechanism of exosomes. What else, exosomes can be used for animal invention by intranasal administration.The purpose of this paper is to help researchers to search more effective drugs and methods of treatment for multiple sclerosis.

免疫耐受在多发性硬化的发病机制中起着重要作用。未成熟树突状细胞（imDC）可以产生调节性T细胞(Treg)，诱导免疫耐受。增强imDC诱导免疫耐受的能力对多发性硬化治疗具有重要意义。本研究应用Treg细胞发育和功能的主要调节基因FoxP3转染imDC，增强其诱导免疫耐受的能力。首先构建FoxP3过表达腺病毒载体，再转染小鼠骨髓源性imDC。提取转染后imDC的外泌体进行研究。外泌体携带其来源imDC的生物学效应，并具有稳定性。验证所提取的外泌体减轻EAE的免疫炎症及促进髓鞘再生的作用。应用蛋白质组学及基因芯片技术分析外泌体所含蛋白质及miRNA成分在转染前后的改变，以及这些改变对免疫炎症及髓鞘再生的作用机制，揭示外泌体对细胞间信号传递的调控。外泌体可经鼻内滴入进行动物干预，研究此种给药途径的生物学效应。该研究项目有助于寻找对多发性硬化更有效的治疗药物及治疗方式。

免疫失衡在多发性硬化的发病机制中起重要作用，T细胞的分化及功能在细胞免疫及分子免疫方面扮演重要角色。树突细胞（DC）作为功能强大的抗原提呈细胞能够调节T细胞的分化。本研究首先构建FoxP3过表达腺病毒载体，再转染不同小鼠的骨髓源性imDC。提取转染FoxP3后imDC的外泌体进行研究。分别对正常小鼠和自身免疫性脑脊髓炎动物模型的小鼠骨髓来源的imDC提取的外泌体进行蛋白质和miRNA的差异分析。并对转染前后的imDC的外泌体进行差异分析。应用不同来源的外泌体对实验性自身免疫性脑脊髓炎动物模型进行体内和体外的实验干预，观察其在免疫炎性调节和髓鞘再生两方面的作用。在免疫调节功能方面应用ELISA、Western blot、qRT-PCR方法检测脾来源免疫细胞及血清免疫因子的变化。FoxP3转染imDC后的外泌体蛋白及miRNA的改变，提示与外泌体所发挥的功能有关。外泌体作为能够携带生物信息的小囊泡有独特的携带优势，可以通过血脑屏障，因此外泌体可经鼻内给予进行动物干预。在比较外泌体不同给予方式对动物模型的影响的同时探寻对多发性硬化更有效的治疗药物及治疗方式。